Thin layer chromatographic determination of aflatoxin in corn dust

Methods adopted by the AOAC and the American Association of Cereal Chemists for determining aflatoxin in corn were modified, and techniques were developed for application to samples of less than 1 to 10 g instead of the specified 50 g samples. Analysis included chloroform extraction of dust samples or dust collected from glass fiber filters, purification of extracts on a silica gel column of appropriate size, and measurement of aflatoxin by either 1- or 2-dimensional thin layer chromatography (TLC). The solvent for 1-dimensional TLC was chloroform-acetone-water (91 + 9 + 1). Solvents for 2-dimensional TLC were, first direction, ether-methanol-water (95 + 4 + 1, lined tank) and second direction, chloroform-acetone-water (91 + 9 + 1, unlined tank), or first direction, chloroform-acetone-water (91 + 9 + 1, unlined tank) and second direction, toluene-ethyl acetate-formic acid (60 + 30 + 10, unlined tank). When samples weighed less than or equal to 0.1 g, the entire concentrated extract was applied to the TLC plate. About 0.5-1.0 ng aflatoxin B1 could be detected on the plate, making the limit of detection about 9 ng/g for 0.1 g samples.     

Laser fluorometric determination of aflatoxin B1 in corn.

A 2-step chromatographic separation, using both thin layer chromatography (TLC) and high pressure liquid chromatography (HPLC), in conjunction with the high sensitivity of laser fluorometry permits extension of the detection limits of aflatoxin contamination in corn to 0.1 ppb (microgram/kg) with a 26% root mean square variation. Aflatoxin B1 is extracted from corn with water-methanol and cleaned up by TLC. The recovery of aflatoxin from the TLC plates was linear from 10 to 1000 pg. Aflatoxin B1 is converted to the more highly fluorescent B2A derivative by treatment with 1N HCl. Experiments with aflatoxin B1 standard establish a constant conversion to B2A over approximately 3 orders of magnitude in B1 concentration. An extract of the B2A aflatoxin derivative is injected onto a reverse phase HPLC column. A flowing droplet of eluant is irradiated by an amplitude-modulated 325 nm He-Cd ion laser beam, and fluorescence from the droplet is detected by a lock-in amplifier in phase with the laser modulation. Several chromatograms are presented that demonstrate the capability of this procedure for removing interfering components in the corn extract.     

Screening method for the detection of aflatoxin, ochratoxin, zearalenone, penicillic acid, and citrinin.

A modification of the official method for ochratoxins and a screening method for zearalenone, aflatoxin, and ochratoxin is described and expanded to include citrinin and penicillic acid. The method uses 0.5N phosphoric acidchloroform (1+10) in the initial extraction; the extract is divided and eluted from 2 columns to provide a quantitative thin layer chromatographic (TLC) method for aflatoxin and ochratoxin in corn and dried beans. Aflatoxin and zearalenone are eluted from one column and ochratoxin, penicillic acid, and citrinin from the other. Ochratoxin A recoveries are low (50%) in peanuts. Zearalenone, penicillic acid, and citrinin were qualitatively recovered from corn and beans; zearalenone and penicillic acid were recovered from peanuts but citrinin was not. Several TLC solvents were used to separate interferences.     

Rapid quantitation and confirmation of aflatoxins in corn and peanut butter, using a disposable silica gel column, thin layer chromatography, and gas chromatography/mass spectrometry

A simple, rapid, and solvent-efficient method for determining aflatoxins in corn and peanut butter is described. Aflatoxins B1, B2, G1, and G2 were extracted from 50 g sample with 200 mL methanol-water (85 + 15). A portion of the extract was diluted with 10% NaCl solution to a final concentration of 50% methanol, and then defatted with hexane. The aflatoxins were partitioned into chloroform. The chloroform solution was evaporated, and the residue was placed on a 0.5 g disposable silica gel column. The column was washed with 3 mL each of hexane, ethyl ether, and methylene chloride. Aflatoxins were eluted with 6 mL chloroform-acetone (9 + 1). The solvent was removed by evaporation on a steam bath, and the aflatoxins were determined using thin layer chromatography (TLC) with silica gel plates and a chloroform-acetone (9 + 1) developing solvent. Overall average recovery of aflatoxin B1 from corn was 82%, and the limit of determination was 2 ng/g. For mass spectrometric (MS) confirmation, aflatoxin B1 in the extract from 3 g sample (20 ng/g) was purified by TLC and applied by direct on-column injection at 40 degrees C into a 6 m fused silica capillary gas chromatographic column. The column was connected directly to the ion source. After injection, the temperature was rapidly raised to 250 degrees C, and the purified extract was analyzed by negative ion chemical ionization MS.     

Comparative evaluation of commercially available aflatoxin test methods

Five qualitative methods and 1 quantitative aflatoxin analytical method were compared with the Holaday-Velasco (HV) minicolumn and thin-layer chromatography (TLC) methods for corn in an evaluation involving 4 U.S. Department of Agriculture Federal Grain Inspection Service (USDA-FGIS) laboratories, 1 laboratory at the University of Georgia, and 1 laboratory at the University of Arizona. Samples analyzed included 1 set of artificially contaminated corn containing both aflatoxin B1 and B2 (ratio of B1:B2 of 92:8), 1 set of artificially contaminated corn containing only aflatoxin B1, and 1 set of naturally contaminated corn. Levels of total aflatoxin tested were 0, 10, 15, 20, 25, 30, and 40 ppb. Results of analysis of these samples with each method evaluated are reported. Chi-square analyses indicated that performance of the Afla-20-Cup, Aflatest, EZ-Screen, OXOID, and SAM-A methods was not statistically different from that of the HV minicolumn. Agri-Screen results were not statistically different from those obtained with TLC.     

Rapid thin layer chromatographic method for determining aflatoxin B1, ochratoxin A, and zearalenone in corn

A multimycotoxin thin layer chromatographic method is described for the analysis of corn. Aflatoxins are extracted from the samples with acetonitrile-water, and sodium bicarbonate is added to separate the acidic ochratoxin from zearalenone and aflatoxin B1. After chloroform extraction, 1N NaOH is added to separate zearalenone and aflatoxin B1. The separated mycotoxins are spotted on TLC plates, which are then examined under ultraviolet light. The following recoveries (%) were obtained for corn samples: aflatoxin B1 71, ochratoxin A 87, and zearalenone 85. The limits of detection for the respective mycotoxins were 2, 40, and 200 ppb.     

An analytical survey of aflatoxins in tissues from swine grown in regions reporting 1988 aflatoxin-contaminated corn.

A joint project was undertaken by the Food Safety and Inspection Service (FSIS) and the Agriculture Research Service branches of the U.S. Department of Agriculture to determine the presence of aflatoxins in the U.S. meat supply during a drought year. In 1988, high incidences of aflatoxins occurred in corn grown in regions of the Midwest, Southeast, and South. Six states were identified as having serious aflatoxin contamination in their corn crop: Virginia, North and South Carolina, Texas, Iowa, and Illinois. Swine liver and pillars of diaphragm (muscle) tissues were sampled by federal FSIS Inspectors in plants located in these states. A worstcase sampling plan was conducted. Samples were taken in January 1989 from hogs fed corn soon after harvest and in April 1989 from hogs fed corn originally stored and then fed in the spring. A modification of the official AOAC method for the thin-layer chromatography (TLC) determination of aflatoxins in animal tissue was used to permit quantitation by LC with fluorescence detection. The official AOAC TLC confirmation of identity method was used to confirm all positive samples with B1 concentrations greater than 0.04 ppb and M1 concentrations greater than 0.1 ppb. Sixty samples in the January group and 100 samples in the April group were assayed. Concentrations of aflatoxins B1 and M1 in the first group of pig livers ranged from 0.04 to 0.06 ppb. The identity of aflatoxin B1 was confirmed in all positive samples. Aflatoxin M1 could not be confirmed in any of the positive liver samples because the method was insufficiently sensitive for this aflatoxin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thin layer chromatographic confirmation of aflatoxin M1 extracted from milk

Aflatoxin M1 can be confirmed directly on a thin layer plate by reacting the toxin with a mixture of reagents containing p-anisaldehyde. This confirmatory procedure requires only 2 elutions in the same direction using 2 different solvents. The mixture containing p-anisaldehyde is overspotted on M1 after the plate has been developed in toluene-ethyl acetate-ethyl ether-formic acid (25 + 35 + 40 + 5). The plate is heated at 110 degrees C for 10 min and then developed in hexane-acetone-chloroform (15 + 50 + 35). The Rf value of the green fluorescent derivative is less than that of the M1 standard. This confirmatory procedure requires only one-dimensional TLC, so several sample extracts and the standard can be run simultaneously. The minimum detectable quantity of aflatoxin M1 on the TLC plate with this test is 0.3 ng. p-Anisaldehyde reagent solution may also be used as a spray reagent for the confirmation of aflatoxin M1. The procedures described were satisfactory for confirming the mycotoxin in spiked samples of powdered and liquid milk.     

Simple, rapid cleanup method for analysis of aflatoxins and comparison with various methods

A method is described for simple and rapid determination of aflatoxins in corn, buckwheat, peanuts, and cheese. Aflatoxins were extracted with chloroform-water and were purified by a Florisil column chromatographic procedure. Column eluates were concentrated and spotted on a high performance thin layer chromatographic (HPTLC) plate, which was then developed in chloroform-acetone (9 + 1) and/or ether-methanol-water (94 + 4.5 + 1.5) or chloroform-isopropanol-acetone (85 + 5 + 10). Each aflatoxin was quantitated by densitometry. The minimum detectable aflatoxin concentrations (micrograms/kg) in various test materials were 0.2, B1; 0.1, B2; 0.2, G1; 0.1, G2; and 0.1, M1. Recoveries of the aflatoxins added to corn, peanut, and cheese samples at 10-30 micrograms/kg were greater than 69% (aflatoxin G2) and averaged 91%, B1; 89%, B2; 91%, G1; 78%, G2; and 92%, M1. The simple method described was compared with the AOAC CB method, AOAC BF method, and AOAC milk and cheese method. These methods were applied to corn, peanut, and cheese composites spiked with known amounts of aflatoxins, and to naturally contaminated buckwheat and cheese. Recoveries were much lower for the BF method compared with our simple method and the CB method.     

Rapid screening method for aflatoxins and zearalenone in corn.

The methanol-water extraction system used in AOAC Method II for aflatoxins extracts both the aflatoxins and zearalenone from corn. Using this methanol-water extraction system as a base, a rapid screening procedure has been developed for these mycotoxins. The methanol-water extract is defatted with hexane and the pigments are precipitated with copper carbonate. The aflatoxins and zearalenone are subsequently extracted into chloroform and are then detected by half-plate TLC. An elapsed time of about 1 hr is required to analyze 1 sample. The sensitivity of the method is about 2 mu-g/kg for aflatoxin B-1 and 100 mu-g/kg for zearalenone.     

High pressure liquid chromatographic determination of aflatoxins in corn.

A high pressure liquid chromatographic (HPLC) method is proposed for determining aflatoxins in corn. The sample is extracted with methanol-10% NaCl (4 + 1), pigments are precipitated with zinc acetate, and the extract is cleaned up on a small (2 g) silica gel column. Aflatoxins in the purified extract are resolved by normal phase HPLC on a microparticulate (10 micrometer) silica gel column with water-saturated chloroform-cyclohexane, acetonitrile solvent, and detected by fluorescence on a silica gel-packed flowcell. The method was compared with chloroform-water extraction of the official CB method on 15 samples of contaminated corn. In 5 of the 6 samples containing aflatoxins B1, B2, G1, and G2, methanol-10% NaCl extracted more aflatoxin than did cloroform-water, as measured both by HPLC and by thin layer chromatography. In samples containing only B1 and B2, the 2 extraction solvents were virtually equivalent. Agreement was good between HPLC and TLC for each extraction solvent. Average recovery of aflatoxins B1, B2, G1, and G2 added to yellow cornmeal at 3 levels was greater than 90%.     

Comparison of postcolumn derivatization-liquid chromatography with thin-layer chromatography for determination of aflatoxins in naturally contaminated corn

Quantitation of aflatoxins by liquid chromatography with postcolumn iodine derivatization (LC-PCD) and fluorescence detection was compared with quantitation by the AOAC CB method, 968.22. Thirty-seven naturally contaminated corn samples were ground and then divided. One portion was extracted, and the extract was cleaned up and analyzed by thin-layer chromatography according to the CB method. The second portion was extracted and cleaned up in a similar fashion, but quantitation was by the LC-PCD method. For aflatoxin B1 concentrations ranging from 0 to 150 ng/g, results obtained by the 2 methods were fitted to a linear equation with the LC-PCD results as the dependent variable. The correlation coefficient was 0.99, the intercept was near 0, and the slope was near 1. For aflatoxin B2, the correlation coefficient was 0.97, and the intercept was near 0. However, the slope of the equation relating LC-PCD concentration to TLC concentration was only 0.5. We believe that this lack of equivalence between the methods for determination of aflatoxin B2 is due to overestimation by the TLC method because the low levels present are near the TLC detection limit for B2.     

Evaluation of enzyme-linked immunosorbent assay of cleanup for thin-layer chromatography of aflatoxin B1 in corn, peanuts, and peanut butter

A simple, rapid enzyme-linked immunoassay (ELISA) was used to evaluate the performance of each step (extraction, filtration, solvent partition, and silica gel column chromatography) of a solvent-efficient thin-layer chromatographic (TLC) method which is undergoing interlaboratory collaborative study for the determination of aflatoxin B1 in corn, raw peanuts, and peanut butter. The apparent average recoveries using the ELISA method were about 30 to 50% higher than those using the TLC method if only the amount of B1 added to the samples was used in the calculations. After the cross-reaction of the antibody with other aflatoxins added to the samples was considered, the amounts recovered approached the levels of aflatoxins added in all 3 commodities tested. With no cleanup treatment, ELISA recoveries at aflatoxin B1 levels above 7.5 ng/g were 84, 79, and 103% for corn, raw peanuts, and peanut butter, respectively. The coefficients of variation were between 5.2 and 25.2%. With each cleanup step in the TLC method, ELISA detected a progressive decrease in recovery from 150.5 to 105.3% (before correction for the presence of other aflatoxins) or from 93.5 to 65.4% (after correction for other aflatoxins) of B1 added to the samples. The ELISA data support the conclusion obtained from previous studies that cleanup treatments were not necessary in the ELISA. When large amounts of other aflatoxins are present, an understanding of the cross-reactivity of antibody with other aflatoxins in the ELISA is essential for final interpretation of the data.     

Enzyme-linked immunosorbent assay for screening aflatoxin B1 in cottonseed products and mixed feed: collaborative study

A joint AOAC/IUPAC (International Union of Pure and Applied Chemistry) interlaboratory study of an enzyme-linked immunosorbent screening assay (ELISA) for aflatoxins was conducted in laboratories in Canada, France, Japan, South Africa, Switzerland, The Netherlands, Tunisia, and the United States. Twenty-eight samples of raw and roasted peanuts, corn, whole cottonseed, cottonseed meal, ammoniated cottonseed meal, and poultry feed containing various quantities of natural aflatoxins and supplemented when appropriate with aflatoxin B1 were distributed to participating laboratories for testing. The assay is based on conjugation of pure aflatoxin B1 to an enzyme and the competition between this conjugate and (free) aflatoxins in the product for aflatoxin-specific antibodies coated onto microtiter well walls. After a wash step to remove all unbound aflatoxins, a substrate, added to each well, is catalyzed from a colorless to a green solution by any bound enzyme-conjugated aflatoxin B1 present. The intensity of the color decreases as the amount of free aflatoxin B1 in the product increases. Overall correlation was good between ELISA and thin-layer chromatographic (TLC) results for cottonseed products and mixed feed. Variable results were reported for corn and peanut product samples. Although some positive samples (greater than 15 ng/g) of cottonseed products and mixed feed were reported to contain less than 15 ng/g by visual determination, a review of data for absorbance measurements showed that the contamination level was close to the greater than or equal to 15 ng/g standard and would not have been reported as negative under routine screening.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thin layer chromatographic determination of citrinin.

A thin layer chromatographic (TLC) method is described for the determination of citrinin in feeds. Citrinin is extracted from feed with methanol and water, the mixture is made alkaline with 10% sodium carbonate, and the aqueous solution is filtered and extracted with chloroform to remove most of the interfering materials. The aqueous layer is acidified with 2N HCl and extracted with chloroform. The chloroform extract is concentrated and spotted on a thin layer chromatographic (TLC) plate which is developed in chloroform-acetone-ethanol-water (60 + 40 + 10 + 1). The citrinin is viewed under ultraviolet light after TLC. Either visual or fluorodensitometric quantitation is used. Recoveries of citrinin from various feed samples spiked at levels of 2.0--5 micrograms/g were 75--92%. The proposed method can detect 0.5 micrograms/g feed, including corn, silage, ready mixed feeds, and feed pellets.     

Fluorimetric determination of aflatoxin M1 in cheese

A simple and sensitive method is proposed for the determination of aflatoxin M1 in cheese. The ground cheese sample is extracted with acetone-water (3 + 1). Acetone is evaporated under vacuum, and the aqueous phase is passed through a C18 disposable cartridge. After the cartridge is washed with acetonitrile-water (1 + 9), the toxin is eluted with acetonitrile. The extract is then cleaned up on a silica cartridge. Final analysis is performed by 2-dimensional thin layer chromatography (TLC) combined with fluorodensitometry or by liquid chromatography on a reverse phase C18 column with fluorescence detection. Recovery is greater than 90%, and the coefficient of variation is 6% or less. The detection limit is in the range of 10 ng/kg. The identity of aflatoxin M1 is confirmed by formation of the M2a or acetyl-M1 derivative and rechromatography.     

Evaluation of a testing program for aflatoxin in corn

A computer model that accounts for sampling and analytical variability was developed to simulate the aflatoxin testing program administered by the North Carolina Department of Agriculture (NCDA) to regulate aflatoxin in corn meal. Monte Carlo solution techniques were employed to account for conditional probabilities that rise from multiple samples being used in the testing program. The NCDA testing program was then evaluated by applying the computer model to a hypothetical group of 1000 corn meal lots with the same distribution of aflatoxin concentrations as was observed among aflatoxin assays made by NCDA on commercial lots of corn meal from 1977 to 1980. The average of the 1000 lots assayed was 17.7 parts per billion (ppb). The model predicted that 79.5% of the lots would be accepted and 20.5% of the lots would be rejected by the NCDA testing program. The accepted and rejected lots contained an average of 5.7 and 64.2 ppb aflatoxin, respectively. The testing program accepted 7.3% of the lots with more than 20 ppb aflatoxin (consumers' risk) and rejected 1.0% of the lots with 20 ppb or less (processors' risk). A correct decision was made 94% of the time.     

A convenient thin layer chromatographic cleanup procedure for screening several mycotoxins in oils.

A thin layer chromatographic cleanup development with benzene-hexane (3+1) effectively removed lipids and some contaminants from mixtures of mycotoxins in corn oil, olive oil, peanut oil, soybean oil, and seed extracts. A second development in the same direction as the first, using toluene-ethyl acetate-formic acid (6+3+1) or benzene-acetic acid (9+1), separated the mycotoxins. Satisfactory separation was achieved for commercial oils spiked with sterigmatocystin, zearalenone, ochratoxins A, B, and C, and aflatoxins B1, B2, G1, and G2. This technique permits detection of 5 ppb aflatoxin B1 in corn.     

Dilution errors in aflatoxin determinations caused by compounds extracted from peanuts

Several methods have been developed to analyze peanuts for aflatoxin by using thin layer chromatography (TLC). These methods depend on solvent extraction of aflatoxin from a sample of the product. Unfortunately, solvent solutions used to extract aflatoxin from peanuts also extract measurable quantities of other compounds such as oils, fats, sugars, and protein. The volume of these extracted compounds causes error in measuring the proportion of the solvent solution analyzed for aflatoxin. Also, because the cleanup procedures for some methods are inadequate, the volume of some of these extracted compounds also causes error in measuring the proportion of the extracted aflatoxin placed on TLC plates. These 2 errors cause underestimation of aflatoxin concentrations by approximately 11, 14, and 5% for the CB method, the modified version of the BF method generally used for raw peanuts, and a water slurry method, respectively. The correction specified by the CB method for fats in the extraction solvent reduces the approximate error for the CB method from 11 to 1%.     

Use of the Mycosep multifunctional cleanup column for liquid chromatographic determination of aflatoxins in agricultural products.

A liquid chromatographic (LC) technique has been developed that uses the Mycosep multifunctional cleanup (MFC) column. MFC columns provide a rapid 1-step extract purification. They are designed to retain particular groups of compounds that may create interferences in analytical methods. At the same time, MFC columns allow compounds of interest to pass through. In the method presented, test samples are extracted in a blender with acetonitrile-water (9 + 1). A portion of the extract is forced through an MFC column designed especially for analysis of numerous mycotoxins. Analytical interferences are retained, while aflatoxins pass through the column. Aflatoxins B1 and G1 are converted to their hemiacetals by heating a mixture of purified extract and water-trifluoroacetic acid-acetic acid (7 + 2 + 1) at 65 degrees C for 8.5 min. An aliquot of this mixture is analyzed by isocratic LC with acetonitrile-water mobile phase and fluorescence detection. A detection limit of less than 0.5 ng/g for aflatoxin B1 was obtained. Average recoveries greater than 95% total aflatoxins (B1, B2, G1, and G2) and coefficients of variation of less than 3% were obtained. The method was successfully applied to the following commodities: corn, almonds, pista-chios, walnuts, peanuts, Brazil nuts, milo, rice, cottonseed, corn meal, corn gluten meal, fig paste, and mixed feeds.